Increase in various protein and RNA-based applications have increased the demand for large-scale production of RNA and protein. Most of these applications, especially therapeutic applications, require the protein/RNA to meet stringent quality criteria in terms of purity, potency, efficacy and safety. Recombinant protein production is often employed to meet the expectation of desired, large scale protein production. Higher production efficiencies and lower costs are much desired to make these protein/RNA products commercially viable.
Traditionally, for in vivo production of protein in mammalian cells, cell transfection using plasmid DNA has been used as a major tool. Production of a desirable protein or RNA from the corresponding DNA sequence involve use of large number of eukaryotic cells and is generally performed in large volume scales such as 100 L. For in vivo protein production, stable cell lines may be generated and expanded to adequate quantities to generate the desired protein yield. Further, in cases where the expressed protein is lethal to the host cells, a transient production system may be used. Significantly higher quantities of RNA or proteins may also be expressed in a shorter period using a cell-free, in vitro RNA or protein expression system. However, the in vitro transcription-translation system often requires larger quantities of a DNA template.
Generally, a plasmid DNA is used as a template DNA in in vivo and in vitro transcription-translation system. However, manufacturing of sufficient plasmid for transient/stable transfection or cell-free expression in larger scale is often expensive and cumbersome. For example, traditional methods of plasmid generation require labor-intensive cloning and plasmid purification. Further, traditional methods of large scale plasmid preparation run the risk of contamination by extraneous bacterial components and/or purification reagents, which may affect the downstream RNA expression and subsequent protein production.
A rapid, cost-effective production of suitable engineered DNA sequences, especially a DNA sequence containing only a promoter and a gene of interest, and devoid of any undesirable DNA sequences that are necessary for maintenance in a bacterium (e.g., origin of replication or an antibiotic resistance gene), which can be used for transfection in eukaryotic cells for subsequent RNA and/or protein production is highly desirable. Isothermal DNA amplification techniques such as rolling circle amplification (RCA) may be employed to generate such large quantities of high-quality DNA with less effort, time, and expense, starting from a circular nucleic acid template. For example, RCA enables rapid production of suitable engineered DNA sequences, for example, a DNA sequence containing only a promoter and a gene of interest.
Though few RNA and protein expression systems using eukaryotic cell lines and the RCA product DNA are currently available, however, those systems suffer from a rather low expression rate of the desired protein, resulting in low yields and high costs of the recombinant protein. There exists a need for an appropriate in vivo RNA and/or protein expression system that provide significant improvements in production of RNA and/or protein that are commercially viable.